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치의과학박사 학위논문
Extracts of Magnoliae Cortex and
Zea mays L. modulate TLR-2
ligand-induced inflammatory
reactions
후박 추출물과 옥수수 불검화 추출물이 TLR-2
ligand로 유도된 염증반응에 미치는 영향
2019년 2월
서울대학교 대학원
치의과학과 치주과학전공
김 재 윤
Abstract
Extracts of Magnoliae Cortex and
Zea mays L. modulate TLR-2
ligand-induced inflammatory
reactions
Jae-Yoon Kim
Program in Periodontology, Department of Dental Science
Graduate School, Seoul National University
(Directed by Professor In-Chul Rhyu, D.D.S, M.S.D., Ph.D.)
Periodontal disease results from undesired immune reactions in
response to periodontal pathogens. The bark of the stems and roots
of Magnolia officinalis (Magnoliae Cortex) and Zea mays L. (maize)
has been found to exhibit pharmacological effects as anti-
inflammatory activity. The aim of this study was to evaluate the
capacity of single and combined applications of Magnoliae Cortex
and maize to modulate inflammation in RAW 264.7 cells stimulated
with TLR-2 ligand. Magnoliae Cortex and/or maize were added to
RAW 264.7 cells, and the cells were stimulated with TLR-2 ligand.
Cytotoxicity and the capacity to modulate inflammation were
determined with a methylthiazol tetrazolium (MTT) assay, nitrite
production, enzyme-linked immunosorbent assay (ELISA), and
western blotting. The statistical analyses were conducted with the
Kruskal-Wallis test, followed by the Mann-Whitney test.
Treatment with Magnoliae Cortex and/or maize inhibited nuclear
factor kappa B (NF-κB) pathway activation and phospho-p44/42
mitogen-activated protein kinase (MAPK) and inducible nitric oxide
synthase (iNOS) protein expression in TLR-2 ligand-stimulated
RAW 264.7 cells. Moreover, the treatments suppressed cytokines
(prostaglandin E2 [PGE2], interleukin [IL]-1β, and IL-6) and
nitrite production. Both Magnoliae Cortex and maize exerted an
anti-inflammatory effect on TLR-2 ligand-stimulated RAW 264.7
cells, and this effect was more pronounced in inhibition of NF-κB
pathway activation and phospho-p44/42 MAPK when the extracts
were combined. These results suggest that combination of
Magnoliae Cortex and Zea mays L. has the potential to inhibit
inflammatory response in periodontal disease.
----------------------------------------
Keywords: inflammation; Magnolia; nuclear factor kappa B; RAW
264.7 cell; TLR-2 ligand; Zea mays
Student number : 2011-30651
Table of Contents
Abstract (in English)
1. Introduction ......................................................................... 1
2. Materials & Methods ........................................................... 5
3. Results ............................................................................... 13
4. Discussion .......................................................................... 28
5. References ......................................................................... 37
Abstract (in Korean) ............................................................. 45
1
Introduction
Periodontal disease results from undesired immune reactions in
response to periodontal pathogens such as Porphyromonas
gingivalis, Prevotella intermedia, and Aggregatibacter
actinomycetemcomitans [1]. An exogenous infection cause local
release of inflammatory mediators such as cytokines, prostaglandin
E2 (PGE2) and metalloproteinases (MMP) [2]. Host-derived
inflammatory mediators cause the pathologic degradation of
collagens and bone resorption in periodontal lesion [2,3]. As the
periodontal lesion deepens, the subgingival biofilm becomes more
anaerobic and the host response becomes more destructive and
chronic [4]. Removing periodontal pathogens by mechanical or
chemical means is a key component of controlling periodontal
disease. The use of host inflammatory modulation agent is an
adjunctive way to treatment of periodontal disease [2]. For instance,
subantimicrobial-dose doxycycline (SDD) suppresses host-
derived MMP and modulates the host immune reaction [3].
Moreover, omega-3 fatty acids (ω-3) and low-dose aspirin (81
mg/day) in addition to scaling and root planning are more effective
slowing periodontal disease progression than scaling and root
planning alone, in patients with moderate to severe periodontitis [5].
2
Patients with aggressive periodontitis have genetic susceptibility of
hyper-inflammatory status regardless of the amount of the
microbial deposit [6]. The modulation of inflammatory mediators
may additionally help to control periodontal disease, along with the
mechanical removal of periodontal pathogens [2].
Macrophage activation is crucial for the progression of multiple
inflammatory diseases through the release of inflammatory
mediators [7]. Inflammatory cytokines and other soluble mediators
are expressed in stimulated macrophages through the activation of
mitogen-activated protein kinase (MAPK) and nuclear factor kappa
B (NF-κB) [8]. The interaction of Toll-like receptors (TLRs) on
macrophages with bacterial components such as lipopolysaccharide
(LPS), lipoproteins, and endotoxin results in the activation of NF-
κB [7,8]. Moreover, P. gingivalis LPS shows TLR-2 agonist
activity rather than TLR-4, therefore TLR-2 ligand could be used
to mimic infection of periodontal pathogens and to activate
macrophage [9]. NF-κB is a key transcription factor for pro-
inflammatory mediators, including cytokines and chemokines such
as interleukin (IL)-1, IL-6, tumor necrosis factor-α (TNF-α),
and PGE2, as well as nitric oxide (NO) [7,10]. In addition, TLR
engagement initiates signal transduction cascades such as
extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun N-
3
terminal kinase 1/2 (JNK 1/2), and p38 MAP kinase pathways [11].
Magnoliae Cortex, the bark of the stems and roots of Magnolia
officinalis, is used to treat acute diarrhea, cramping abdominal pain,
regurgitation, vomiting, and dyspepsia [12]. Honokiol and magnolol
are phenolic compounds that have been isolated from Magnoliae
Cortex and are known to suppress the production of LPS-mediated
cellular responses such as TNF-α, PGE2, and NO expression [13].
Honokiol inhibits TNF-α and IL-6 in a dose-dependent manner
[14]. Additionally, honokiol has anti-inflammatory effects on
activated macrophages by inhibiting TNF-α and NO expression
through inhibition of the MAPK, protein kinase C-α (PKC-α), and
NF-κB pathways [15]. Magnolol inhibits the IL-6-induced Janus
kinase (JAK)/signal transduction and activator of transcription
(STAT) 3 signaling pathway by reducing STAT3 binding activity in
endothelial cells [16].
The corn silk and corn kernels of Zea mays L. (maize) contain
zeatin, flavonoids, alkaloids, allantoin, saponins, volatile oils,
vitamins, starch, fats, cellulose, and β-sitosterol [17]. Corn silk
and corn kernels have been found to exhibit pharmacological effects,
including anti-hepatoma and anti-fatigue properties [18,19]. Corn
bran inhibits NO production and inducible nitric oxide synthase
(iNOS) expression in a dose-dependent manner [20]. Moreover, a
4
maize husk extract shows anti-inflammatory properties which
inhibit acute and chronic inflammatory mediators in vivo [21]. In
addition, an unsaponifiable fraction of maize reduces gingival
inflammation and tooth mobility in patients with periodontal disease
[22]. An unsaponifiable fraction of maize also reduces probing
depth in patients with oral hygiene instruction [23], however there
is no significant difference with control in patients with periodontal
surgery [23, 24]. In previous studies, maize and Magnoliae Cortex
promote bone tissue regeneration in rats [25], and facilitate clinical
improvement in a dog model of experimental periodontitis [26].
However, the underlying mechanisms have not yet been established.
It is possible to combine more than one substance to modulate
multi-targeted inflammation processes and functions. In such cases,
the combination can produce stable and synergistic effects. The aim
of this study was to evaluate the capacity of Magnoliae Cortex and
maize to modulate inflammation in RAW 264.7 cells stimulated with
TLR-2 ligand. Magnoliae Cortex and maize were separately or
simultaneously applied to cells, and the induced inflammatory
reactions were measured as the amount of NO, PGE2, IL-1β, IL-6,
phospho-p44/42 MAPK, iNOS, and NF-κB produced.
5
Material and Methods
Sample preparation
The soft 75% ethanol Magnoliae Cortex extract was provided by
Dongbang FTL (Seoul, Korea). The extract was produced by
Dongbang FTL as follows. Magenoliae Cortex was ground into fine
particles, and reflux was performed with 75% ethanol at 65°C for
30 minutes. The sample was filtered, and the residue was subjected
to reflux with 75% ethanol at 65°C for 30 minutes; the sample was
filtered. The two filtrates were mixed and subsequently evaporated
under vacuum to obtain the soft extract. The titrated, unsaponifiable
maize extract fraction was provided by Dongkook Pharmaceutical
Co., Ltd. (Seoul, Korea). Briefly, corn oil was mixed with ethanol
and sodium hydroxide, and reflux was performed at 80 °C for 3
hours. The samples were then cooled to room temperature (RT)
and filtered under vacuum. The residue was extracted with 75%
ethanol at 80°C and filtered. The two filtrates were mixed and
concentrated to 1/3 of the volume at 80°C and then cooled to RT.
The samples were subsequently extracted with ethyl acetate,
followed by addition of potassium hydroxide and washing with
purified water. The samples were concentrated to 1/3 of the volume
at 80 °C, and then cooled to RT. Next, ethanol was added, followed
6
by mixing and evaporation under vacuum to obtain the titrated
unsaponifiable maize extract fraction. Ibuprofen (Sigma-Aldrich, St.
Louis, MO, USA) was used as a positive control. Dimethyl sulfoxide
(DMSO) was used as a solvent. The final soft 75% ethanol
Magnoliae Cortex extract concentration was 60 μg/mL in 1%
DMSO; this was denoted as M. The final concentration of the
titrated unsaponifiable maize extract fraction was 300 μg/mL in 1%
DMSO; this was denoted as Z. The combined treatment of M and Z
was denoted as MZ. The final ibuprofen concentration was 10 mM
in 1% DMSO; this was denoted as IBU.
Cell culture
RAW 264.7 cells (murine) were obtained from Korean Cell Line
Bank (KCLB, Seoul, Korea) and cultured in a 5% CO2 atmosphere at
37°C in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand
Island, NY, USA) containing 10% fetal bovine serum (FBS;
Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin
(Invitrogen) (complete DMEM). The cells were inoculated in 10-
cm dishes at a density of 1.9×104/cm2, 24-well plates at a density
of 5×104/cm2, and 96-well plates at a density of 3.3×104/cm2 and
7
cultured for 24 hours. The cells were pre-treated with M, Z or MZ,
with or without Pam3CSK4 (InvivoGen, San Diego, CA, USA), a
synthetic TLR-2 ligand. IBU was used as a positive control.
Cell viability measurements using the MTT assay
RAW 264.7 cells were inoculated into a 96-well plate and
incubated for 24 hours in complete DMEM. The culture medium was
subsequently discarded and replenished as follows: with medium
alone; with the soft 75% ethanol Magnoliae Cortex extract (0.6, 6
or 60 μg/mL); with the titrated, unsaponifiable maize extract
fraction (3, 30 or 300 μg/mL); or with a mixture of one of the
extracts and IBU (10 mM) for 24 hours. Filtered methylthiazol
tetrazolium (MTT) solution in DMEM was added to each well (5 mg
MTT/mL), and the cells were incubated at 37°C for 4 hours. The
unreacted dye was then removed. The insoluble MTT formazan
crystals were allowed to dissolve in DMSO at room temperature for
15 minutes, and absorbance was measured at 570 nm using a
microplate reader (Molecular Devices, Sunnyvale, CA, USA).
8
Nitrite production measurements
RAW 264.7 cells were inoculated into 24-well plates and
incubated for 24 hours in complete DMEM as follows: with medium
alone; with medium and Pam3CSK4 (10 μg/mL); with the soft 75%
ethanol Magnoliae Cortex extract (0.6, 6, or 60 μg/mL) and
Pam3CSK4 (10 μg/mL); with the titrated, unsaponifiable maize
extract fraction (3, 30, or 300 μg/mL) and Pam3CSK4 (10
μg/mL); with a mixture of one of the extracts and Pam3CSK4 (10
μg/mL); or with IBU (10 mM) and Pam3CSK4 (10 μg/mL). To
each well, 80 μL of Griess reagent (1% sulfanilamide in 5%
phosphoric acid and 0.1% N-[1-naphthyl] ethylenediamine
dihydrochloride in water; Sigma-Aldrich) was added, and the
mixture was incubated for 5 minutes in the dark. The total nitrite
level was measured and calculated on the basis of absorbance at
540 nm using a microplate reader (Molecular Devices).
ELISA for PGE2, IL-1β, and IL-6 release
measurements
Cytokines were measured by enzyme-linked immunosorbent
assay (ELISA). RAW 264.7 cells were plated in a 24-well plate and
9
cultured in DMEM containing 10% FBS for 24 hours. These cells
were then serum-starved overnight in 0.5% FBS-containing
medium. For PGE2 release measurement, the cells were pretreated
for 30 minutes before treatment with Pam3CSK4 (10 μg/mL) for
24 hours in a 5% CO2 incubator at 37°C with medium alone; with
the soft 75% ethanol Magnoliae Cortex extract (6 or 60 μg/mL);
with the titrated, unsaponifiable maize extract fraction (30 or 300
μg/mL); or with a mixture of one of the extracts and IBU (10 mM).
The collected media were stored at −70°C for cytokine analysis.
The supernatant was assessed using PGE2-specific ELISA kits
(R&D system, Minneapolis, MN, USA) according to the
manufacturer’s instructions. The absorbance was read in a
microplate reader (Molecular Devices). For IL-1β and IL-6
release measurements, the cells were pretreated with M (60
μg/mL), Z (300 μg/mL), MZ (60 μg/mL M and 300 μg/mL Z), or
IBU (10 μg/mL) for 30 minutes prior to treatment with Pam3CSK4
(10 μg/mL) for 24 hours in a 5% CO2 incubator at 37°C. The
collected media were stored at −70°C for cytokine analysis. The
supernatant was evaluated using a bead-based multiplex cytokine
assay for IL-1β and IL-6 (Milliplex, Millipore, Billerica, MA,
USA) and analyzed with a Luminex 200 System (Luminex, Austin,
TX, USA).
10
iNOS, COX2, and phospho-p44/42 MAPK
measurements by western blotting
RAW 264.7 cells were plated in 10-cm dishes and cultured in
DMEM containing 10% FBS for 24 hours and then serum-starved
overnight in 0.5% FBS medium. The cells were pretreated with M
(60 μg/mL), Z (300 μg/mL), MZ (60 μg/mL M and 300 μg/mL
Z), or IBU (10 μg/mL) for 30 minutes prior to treatment with
Pam3CSK4 (10 μg/mL) for 24 hours (iNOS and COX-2) or 30
minutes (phospho-p44/42 MAPK) in a 5% CO2 incubator at 37°C.
The cells were washed with ice-cold phosphate-buffered saline
and harvested with protein extraction solution (50 mM Tris-HCl,
pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, and 1 mM PMSF)
(Elpisbio, Daejeon, Korea) according to the manufacturer’s
protocol. Total cell lysates (10 μg) were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred onto nitrocellulose membranes. The membranes
were blocked with 5% bovine serum albumin (BSA) for 1 hour, and
primary antibodies against iNOS (Cell Signaling Technology,
Danvers, MA, USA), cyclooxygenase-2 (COX-2; Cell Signaling
11
Technology), phospho-ERK1/2 (Cell Signaling Technology),
ERK1/2 (Cell Signaling Technology), and β-actin (Sigma-
Aldrich) were added to the Tris-buffered saline (TBS)-T solution
containing 5% BSA and incubated overnight at 4°C. After 3 washes
with TBS-T buffer, the membranes were incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell
Signaling Technology) in TBS-T-containing 5% BSA for 1 hour.
The membranes were subsequently washed 3 times with TBS-T
buffer and analyzed with enhanced chemiluminescence detection
reagents (Dogen, Innotech, Daejeon, Korea).
NF-κB activation measurements
RAW 264.7 cells were plated in 10-cm dishes and cultured in
DMEM containing 10% FBS for 24 hours and then serum-starved
overnight in 0.5% FBS medium. The cells were pretreated with M
(60 μg/mL), Z (300 μg/mL), MZ (60 μg/mL M and 300 μg/mL
Z), or IBU (10 μg/mL) for 30 minutes prior to treatment with
Pam3CSK4 (10 μg/mL) for 30 minutes in a 5% CO2 incubator at
37°C. NF-κB activation was measured using an NF-κB p65
ActiveELISA kit (Imgenex Corp, San Diego, CA, USA) according to
the manufacturer's instructions. Briefly, the cells were washed with
12
ice-cold phosphate-buffered saline and lysed with hypotonic buffer
and 10% detergent solution (Imgenex Corp) at 4°C. The
supernatant was collected for cytosolic NF-κB p65. The nuclear
pellets were subsequently lysed with nuclear lysis buffer (Imgenex
Corp), and the supernatant were stored at −80°C for nuclear NF-
κB p65. The supernatants of cytosolic NF-κB p65 were combined
with the supernatants of nuclear NF-κB p65 for total NF-κB p65
analysis. Absorbance at 405 nm was determined using a microplate
reader (Molecular Devices).
Statistical analysis
Data were analyzed using SPSS for Windows version 12.0 (SPSS
Inc., Chicago, IL, USA). The results are expressed as
means±standard deviations. The statistical analyses were
conducted with the Kruskal-Wallis test, followed by the Mann-
Whitney test. Differences were considered to be significant when
the P values were <0.05.
13
Results
Preliminary experiments to determine concentrations
and the mixture ratio of M and Z
In preliminary experiments, various concentrations of the
Magnoliae Cortex extract, the maize extract and ibuprofen were
examined with MTT assay and NO production measurement. The
optimal concentrations with minimal cytotoxic effects and maximum
inhibition of NO production were 60 μg/mL for M, 300 μg/mL for
Z and 10 mM for IBU (data not shown). Nuclear NF-κB p65 were
measured with various mixture ratios of the Magnoliae Cortex
extract and the maize extract, concentrations with minimal
cytotoxic effects, to determine the mixture ratio. The optimal
mixture ratio of Magnoliae Cortex extract and the maize extract
was 1: 5 (60 μg/mL of M : 300 μg/mL of Z; data not shown).
Cytotoxicity of M and Z treatments in RAW 264.7
cells
Concentrations of 0.6, 6 and 60 μg/mL of the Magnoliae Cortex
extract and/or 3, 30 and 300 μg/mL of the maize extract and 10
14
mM of ibuprofen exhibited similar cell viability with control group,
and there were no significant difference (Figure 1).
Figure 1. Cytotoxicity of M and Z treatments in RAW 264.7 cells.
The data are expressed as mean±standard deviation, as obtained
from 6 separate experiments.
M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1%
DMSO), Z: titrated unsaponifiable maize extract fraction (300
μg/mL in 1% DMSO), IBU: ibuprofen (10 mM in 1% DMSO),
DMSO: dimethyl sulfoxide, MZ: the combination treatment of M and
Z.
15
Effects of M and Z on Pam3CSK4-induced NO
production by RAW 264.7 cells
To evaluate the potential anti-inflammatory effects of M and Z
on Pam3CSK4-stimulated RAW 264.7 cells, NO production was
measured. Pam3CSK4 treatment alone dramatically induced NO
production in comparison with the control group, whereas the M and
Z treatments significantly suppressed NO production in
Pam3CSK4-induced RAW 264.7 cells (Figure 2). Concentrations of
0.6 μg/mL of the Magnoliae Cortex extract and 3 μg/mL of the
maize extract and combination of 0.6 μg/mL of the Magnoliae
Cortex extract and 3 μg/mL of the maize extract displayed similar
NO production with Pam3CSK4 group, and there were no significant
difference. However, combination of 6 μg/mL of the Magnoliae
Cortex extract and 30 μg/mL of the maize extract significantly
suppressed NO production compare with Pam3CSK4 group,
meanwhile single treatment of 6 μg/mL of the Magnoliae Cortex
extract and 30 μg/mL of the maize extract showed no significant
difference with Pam3CSK4 group. Z significantly inhibited NO
production compare with Pam3CSK4 group, also significantly
difference with IBU. M and MZ significantly inhibited NO production
compare with Pam3CSK4 group, exhibited no significant difference
16
with IBU (Figure 2).
Figure 2. Effects of M and Z on Pam3CSK4-induced NO production
in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ,
or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In
experiments without Pam3CSK4, the medium alone was used as a
negative control, and IBU was used as a positive control. The data
are expressed as mean±standard deviation, as obtained from 6
separate experiments.
Pam3CSK4: synthetic Toll-like receptor-2 ligand, NO: nitric oxide
a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison
with the IBU
17
Effects of M and Z on PGE2, IL-1β, and IL-6
production in Pam3CSK4-induced RAW 264.7 cells
To determine the effects of M and Z on PGE2 formation, cells
were treated with M, Z, MZ, or IBU for 30 minutes and then
stimulated with 10 μg/mL of Pam3CSK4 for 24 hours. PGE2 was
significantly induced in Pam3CSK4-stimulated cells. However, the
addition of M and Z significantly suppressed PGE2 production in
Pam3CSK4-induced RAW 264.7 cells. Moreover, M and Z inhibited
PGE2 production in a manner similar to that of IBU treatment, and
there were no significant difference. Combined application of 6
μg/mL of the Magnoliae Cortex extract and 30 μg/mL of the maize
extract and single application of 6 μg/mL of the Magnoliae Cortex
extract significantly suppressed PGE2 production compare with
Pam3CSK4 group, meanwhile single treatment of 30 μg/mL of the
maize extract showed no significant difference with Pam3CSK4
group (Figure 3).
18
Figure 3. Effects of M and Z on Pam3CSK4-induced PGE2
formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with
M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL
Pam3CSK4 for 24 hours at 37°C. The data are expressed as
mean±standard deviation, as obtained from 6 separate experiments.
PGE2: prostaglandin E2
a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison
with the IBU
19
According to our results, Pam3CSK4 significantly elevated IL-
1β and IL-6 levels in RAW 264.7 cells (Figures 4 and 5), but IL-
1β and IL-6 production was strongly reduced when the cells were
treated with M and Z. Treating the cells with M and MZ significantly
suppressed IL-1β production compare with Pam3CSK4, and there
were significant difference with Z and IBU (Figure 4). This result
shows that with regard to IL-1β, M treatment inhibited
inflammation more effectively than Z. However, Z and MZ
treatments more significantly inhibited IL-6 levels than M and IBU
(Figure 5), indicating that with regard to IL-6, Z was more efficient
at suppressing inflammation, even when considering the possibility
of a synergistic effect of MZ on IL-6 suppression. Interestingly,
IBU treatment promoted IL-6 production to a significantly greater
degree than Pam3CSK4 treatment alone (Figure 5).
20
Figure 4. Effects of M and Z on Pam3CSK4-induced IL-1β
formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with
M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL
Pam3CSK4 for 24 hours at 37°C. The data are expressed as
mean±standard deviation, as obtained from 6 separate experiments.
IL-1β: interleukin-1β
a)P<0.05 in comparison with Pam3CSK4; b) P<0.05 in comparison
with IBU.
21
Figure 5. Effects of M and Z on Pam3CSK4-induced IL-6
formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with
M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL
Pam3CSK4 for 24 hours at 37°C. The data are expressed as
mean±standard deviation, as obtained from 6 separate experiments.
IL-6: interleukin-6
a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison
with the control
22
Effects of M and Z on p44/42 MAPK activation in
Pam3CSK4-induced RAW 264.7 cells
To assess the effects of M and Z on p44/42 MAPK activation,
cells were treated with M, Z, MZ, or IBU for 30 minutes and then
stimulated with 10 μg/mL of Pam3CSK4 for 30 minutes.
Pam3CSK4 treatment led to significant increases in the levels of
phospho-p44/42 MAPK; however, MZ inhibited p44/42 MAPK
activation in Pam3CSK4-induced RAW 264.7 cells (Figure 6A and
B). M, Z and IBU group had similar levels of phospho-p44/42
MAPK with Pam3CSK4 group, and there were no significant
difference (Figure 6A and B).
23
Figure 6. Effects of M and Z on phospho-p44/42 MAPK expression
levels in Pam3CSK4-induced RAW 264.7 cells. The levels of
phospho-p44/42 MAPK and β-actin expression were detected by
western blotting using specific antibodies. Cells (1.9×104/cm2)
were treated with M, Z, MZ, or IBU for 30 minutes before being
treated with 10 μg/mL Pam3CSK4. To detect phospho-p44/42
MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C.
The blots shown here are representative of 3 independent
experiments. Each sample contained 10 μg of total protein. (A)
The levels of p44/42 MAPK, phospho-p44/42 MAPK and β-actin
expression; (B) ratio of phospho-p44/42 MAPK to p44/42 MAPK
expressed as a percentage.
MAPK: mitogen-activated protein kinase
a)P<0.05 in comparison with Pam3CSK4
24
Effects of M and Z on iNOS, and COX-2 expression in
Pam3CSK4-induced RAW 264.7 cells
To evaluate the effects of M and Z on iNOS and COX-2
expression, cells were treated with M, Z, MZ, or IBU for 30 minutes
and then stimulated with 10 μg/mL of Pam3CSK4 for 24 hours.
iNOS expression increased after Pam3CSK4 stimulation in RAW
264.7 cells, however M, Z, MZ, and IBU significantly inhibited iNOS
expression (Figure 7A and B). There was no change in COX-2
expression following treatment with Pam3CSK4, M, Z, MZ and IBU
(Figure 7A).
25
Figure 7. Effects of M and Z on nuclear iNOS and COX-2
expression levels in Pam3CSK4-induced RAW 264.7 cells. The
levels of iNOS, COX-2, and β-actin expression were detected by
western blotting using specific antibodies. Cells (1.9×104/cm2)
were treated with M, Z, MZ, or IBU for 30 minutes before being
treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2
expression, Pam3CSK4 was added for 24 hours at 37°C. The blots
shown here are representative of 3 independent experiments. Each
sample contained 10 μg of total protein. (A) The levels of iNOS,
COX-2, and β-actin expression; (B) ratio of iNOS to β-actin
expressed as a percentage.
iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2
a)P<0.05 in comparison with Pam3CSK4
26
Effects of M and Z on Pam3CSK4-induced NF-κB
transactivation in RAW 264.7 cells
To determine whether M and Z could affect the level of p65
expression in RAW 264.7 cells, cells were treated with M, Z, MZ, or
IBU for 30 minutes and then stimulated with 10 μg/mL of
Pam3CSK4 for 2 hours. M significantly reduced the level of p65
compare with Pam3CSK4 group (Figure 8). Moreover, MZ
significantly inhibited NF-κB transactivation to the control level,
and there were significant difference with IBU (Figure 8). However,
Z and IBU had similar level of p65 with Pam3CSK4 group, and there
were no significant difference (Figure 8). These results show a
synergistic effect of M and Z on Pam3CSK4-induced RAW 264.7
cells with respect to NF-κB inhibition.
27
Figure 8. Effects of M and Z on Pam3CSK4-induced NF-κB
transactivation in RAW 264.7 cells. Cells (5×104/cm2) were treated
with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10
μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as
mean±standard deviation, as obtained from 6 separate experiments.
NF-κB: nuclear factor kappa B, p65: transcription factor p65.
a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison
with IBU
28
Discussion
The primary etiology of periodontal disease is infection by
periodontal pathogens such as P. gingivalis, P. intermedia, and A.
actinomycetemcomitans [1-3,27]. Conversely, periodontal disease
progresses in response to the host immune inflammatory reaction to
periodontal pathogens and their products [2,3,27]. TLR-2 ligands
from periodontal pathogens stimulate immune cells, such as
neutrophils, monocytes, and macrophages, and immune cells
produce cytokines by multiple processes [2,28]. Degradation action
on collagen fibers is performed by MMPs released by host cell that
responds to the inflammatory cytokines [6,29]. Therefore, the
inflammatory mediators, such as IL-1β, IL-6, and PGE2, cause
local tissue destruction and alveolar bone loss [2,3,27].
Aggressive periodontitis generally involves rapid attachment loss
and bone destruction regardless of the amount of the microbial
deposit [6,27,30]. Single nucleotide polymorphisms (SNP) on genes
encoding cytokines, receptors, metabolic modulators and proteins
result in increased disease susceptibility or overexpression of the
host innate inflammatory reaction [27]. The local inflammatory
reaction by the microbial deposit, overexpressed by hyper-
responsiveness of inflammatory mediators, resulted in local tissue
29
destruction in the host with these SNPs [27].
Modulators of inflammatory mediators, such as SDD, suppress
host-derived MMPs in the periodontal lesion and inhibit local tissue
destruction [2,3]. Waite et al. reported that the patients taking
non-steroidal anti-inflammatory drugs had significantly shallower
depths of periodontal pockets [31]. El-Sharkawy et al. published
that ω-3 and low dose of aspirin adjunctive with scaling and root
planning significantly reduced probing depths compared with scaling
and root planning in patients with moderate to severe periodontitis
[5]. In the model of experimental periodontitis in non-human
primates, IL-1 and TNF antagonist inhibited loss of tissue
attachment [32]. In the model of experimental periodontitis in
beagle dogs, subcutaneous injections of IL-11, known to
downregulate inflammatory mediators, reduced attachment loss and
bone resorption [33]. The modulators of inflammatory mediators
may be beneficial for slowing the progression of periodontal disease,
especially aggressive periodontitis, along with the mechanical
removal of oral pathogens [2-4].
Magnolol and honokiol, the main components of Magnoliae Cortex,
have been reported to suppress inflammatory reactions both in vitro
and in vivo [15,34]. One of the main components of maize, β-
sitosterol, has been shown to exhibit anti-inflammatory effects that
30
suppress monocyte activation by IL-6 and TNF-α and might be
useful as a treatment for rheumatoid arthritis [35]. Although anti-
inflammatory effects of Magnoliae Cortex and maize have been
presented in previous studies, details regarding the mechanism
through which these effects occur have not been reported [20-
22,34]. Herein, we demonstrated that Magnoliae Cortex and/or
maize can modulate the inflammatory response by suppressing
TLR-2 ligand-induced signaling and pro-inflammatory molecule
production.
NO, an inflammatory mediator, is a reactive molecule that has a
variety of effects depending on its relative concentration [36].
Small amounts of NO are produced by constitutive nitric oxide
synthase (NOS) enzymes (i.e., endothelial NOS and neural NOS),
and are essential for physiological homeostasis [36]. In contrast,
the large amounts of NO produced by inducible NOS have been
closely linked to the pathophysiology of numerous inflammatory
diseases [36,37]. In the model of experimental periodontitis in rat,
ligation caused a significant increase in the iNOS activity [38].
However, the treatment of a selective inhibitor of iNOS resulted in a
significant inhibition of alveolar bone loss [38]. In the present study,
the high level of NO production observed in Pam3CSK4-stimulated
macrophages was strikingly suppressed by M and Z, with an
31
effectiveness that was almost the same as that of IBU. NO inhibition
by iNOS downregulation in macrophages modulates host immune
inflammation induced by TLR-2 ligand, and the results presented
herein correspond well with those of earlier studies. For example,
previous studies demonstrated that honokiol and magnolol, the
major active components of Magnoliae Cortex, inhibited NO release
and iNOS expression in a dose-dependent manner [13,15,34]. In
addition, a study of corn bran showed that 80% of an ethanolic corn
bran extract inhibited NO production and iNOS expression in
macrophages stimulated with bacterial products [20].
The NF-κB pathway, which is considered to be a prototypical
pro-inflammatory signaling cascade, is activated by TLRs that
recognize microbial molecular patterns [39]. NF-κB binds to the
iNOS gene promoter, thereby promoting iNOS expression and NO
production [36]. However, some drugs, such as salicylates, only
inhibit iNOS expression without affecting NF-κB-mediated iNOS
mRNA expression [40]. In the present study, M and MZ inhibited
Pam3CSK4-induced NF-κB activation, whereas Z had similar
level of NF-κB activation with Pam3CSK4 group. These findings
are different from those of Kim et al., who reported that corn
phenolic amides contributed to the inhibition of NO production by
downregulating the level of NF-κB-mediated iNOS gene
32
expression [20]. Moreover, Rho et al. also reported that Zea mays
husk extract inhibited NO production by suppressing the level of
NF-κB-mediated iNOS gene expression [41]. These results
indicate that the various components of corn interact with
inflammatory processes via different pathways. In the present study,
MZ significantly suppressed NF-κB activation, and this activity
was more pronounced than that of M, suggesting that M and Z
exerted a synergistic effect with respect to NF-κB inhibition.
In addition to the NF-κB signaling pathway, JAK-STAT
signaling regulates inflammatory processes [36,42]. Cytokines such
as interleukins and interferons activate the Janus family of tyrosine
kinases (JAK1, JAK2, JAK3, and tyrosine kinase 2 [Tyk2]), and
activated JAK kinases phosphorylate members of the STAT family
[42]. In addition to interleukins and interferons, other cytokines,
such as IL-6, also activate JAK1 and STAT3 [16]. Suppression of
IL-6-mediated JAK-STAT signaling is important for controlling
inflammation [16]. In the present study, M and Z significantly
suppressed Pam3CSK4-induced IL-6 production in RAW 264.7
cells, and Z was more effective at inhibiting IL-6 secretion than
was M. These results are assumed that Z does not suppress the
NF-κB pathway, but instead suppresses the JAK-STAT pathway,
and that M suppresses both pathways to exert its anti-
33
inflammatory effects. These results are consistent with those of an
earlier study reporting that magnolol extracted from Magnoliae
Cortex suppressed IL-6-induced STAT3 phosphorylation and
downstream target gene expression in endothelial cells [16].
Interestingly, the results of present study show that IBU
significantly increased Pam3CSK4-induced IL-6 production, but
not IL-6 production without Pam3CSK4 stimulation (data not
shown). These findings correspond well with those of earlier
studies, in which ibuprofen significantly increased Pam3CSK4-
induced IL-6 production [43] but had no inhibitory effects on IL-6
bioactivity [44]. Although IBU suppressed inflammation, further
studies are needed to reveal the precise mode of action with regard
to inflammatory suppression.
Parallel to the NF-κB and JAK-STAT pathways, the ERK 1/2
pathway is also key for regulating inflammation at the
transcriptional level. Recognition of bacterial components by TLRs
leads to the phosphorylation of interleukin-1 receptor-associated
kinase (IRAK), which in turn activates tumor necrosis factor
receptor-activated factor 6 (TRAF6) [8]. TRAF6 activates IκB
kinase and MAPK, which phosphorylate downstream kinases.
Activated IκB kinase and MAPK then activate p38 and ERK 1/2 as
well as NF-κB, leading to IL-1β, TNF-α, and NO transcription
34
and secretion [8]. In the present study, MZ significantly reduced
phospho-p44/42 MAPK expression in Pam3CSK4-stimulated
macrophages, but neither M nor Z could inhibit the ERK 1/2
pathway alone. These results indicate that M and Z exerted a
synergistic effect in inhibiting the ERK 1/2 pathway, suggesting that
they may control inflammation more effectively when used together.
The increased release of active IL-1β is a hallmark of auto-
inflammatory diseases and chronic inflammatory diseases. IL-1β
entering the circulation from a local inflammation site induces IL-1
itself as well as systemic inflammation [45,46]. Both IL-1β bursts
and IL-1β polymorphisms are closely related to the slowly
progressive inflammatory processes that occur in periodontitis,
coronary heart disease, osteoarthritis, and type 2 diabetes [45-47].
As a result, blocking IL-1β has been the focus of auto-
inflammatory and chronic inflammatory disease treatments [46]. In
the present study, M and Z efficiently blocked IL-1β secretion in
Pam3CSK4-stimulated RAW 264.7 cells. Additionally, M
significantly and more effectively blocked IL-1β secretion than
IBU. These results suggest that M and Z can modulate not only
chronic inflammatory reactions but also auto-immune inflammatory
reactions.
35
Figure 9. Schematic model for the anti-inflammatory mechanism of
M and Z in Pam3CSK4-induced RAW 264.7 cells. ERK 1/2:
extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear
factor kappa B pathway, JAK/STAT3: Janus kinase/signal
transduction and activator of transcription 3 signaling pathway
36
In conclusion, both the Magnoliae Cortex and maize extracts
modulated the downstream targets of multiple inflammatory
pathways, and decreased TLR-2 lignad-induced inflammatory
reactions by inhibiting NO, PGE2, phospho-ERK 1/2, iNOS, IL-1β,
IL-6, and NF-κB levels. However, a difference in their modes of
action was observed, in that Magnoliae Cortex was more effective
at decreasing NO, IL-1β, and NF-κB levels, while maize was
more effective at reducing IL-6 levels. In all experiments, the
combination of Magnoliae Cortex and maize showed either the same
or enhanced results relative to the use of Magnoliae Cortex or
maize alone. Additionally, greater efficacy was obtained with
respect to NF-κB and phospho-ERK 1/2 inhibition when both
extracts were added simultaneously (Figure 9). These results
suggest that combination of Magnoliae Cortex and maize has the
potential to inhibit inflammatory response in periodontitis. Further
study is needed to elucidate the precise anti-inflammatory signaling
pathways targeted by each of the components of Magnoliae Cortex
and maize.
37
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45
국문초록
후박 추출물과 옥수수 불검화
추출물이 TLR-2 ligand로 유도된
염증반응에 미치는 영향
김 재 윤
서울대학교 대학원 치의과학과 치주과학전공
(지도교수 류인철)
치주질환은 치주원인균의 자극에 의한 염증반응에 의해 시작된다. 후
박과 옥수수는 예전부터 염증조절 용도로 많이 사용되어 왔고, 최근에
다양한 연구들이 이를 뒷받침하고 있다. 이 연구의 목적은 TLR-2
ligand에 의해 자극된 RAW 264.7 세포에 후박 추출물 및 옥수수 불검
화 추출물을 각각 또는 함께 적용하였을 때 염증반응의 조절능력을 알아
보는 것이다. RAW 264.7 세포에 후박추출물 및 옥수수 불검화 추출물
46
을 각각 또는 함께 처리한 상태에서 TLR-2 ligand로 세포를 자극시켰
다. 각 추출물의 세포독성과 염증조절능력을 세포독성실험
[methylthiazol tetrazolium (MTT) assay], 아질산염생성측정법
(nitrite production), 효소면역분석법 (ELISA; enzyme-linked
immunosorbent assay) 및 웨스턴 블롯 (western blot) 등으로 분석했
다. RAW 264.7 세포에 후박 추출물 및 옥수수 불검화 추출물을 각각
또는 함께 처리 후 TLR-2 ligand로 자극했을 때, 각 추출물은 RAW
264.7 세포의 nuclear factor kappa B (NF-κB) 경로 활성과 인산화
된 p44/42 mitogen-activated protein kinase 그리고 inducible nitric
oxide synthase (iNOS) 단백질 생성을 억제했다. 또한 각 추출물은
RAW 264.7 세포의 사이토카인들 (prostaglandin E2 [PGE2],
interleukin [IL]-1β, and IL-6) 과 아질산염 생성을 억제했다. 후박
및 옥수수 불검화 추출물 모두 TLR-2 ligand에 의해 유도된 RAW
264.7 세포의 염증반응을 억제하였고, 두 추출물을 함께 처리하였을 때
NF-κB 경로 활성 억제 및 인산화된 p44/42 MAPK 억제에 더 효과적
이었다. 이러한 결과는 후박 및 옥수수 불검화 추출물이 치주질환의 염
증반응을 억제할 수 있다는 가능성을 보여준다.
----------------------------------------
주요어 : 염증; 후박; nuclear factor kappa B; RAW 264.7 세포; TLR-
2 ligand; 옥수수
학번: 2011-30651